qHTS for Inhibitors of Polymerase Iota

DNA polymerases, such as Pol iota, are specifically involved in DNA repair. Pol iota specifically plays an important role in translesion synthesis, where the normal high-fidelity DNA polymerases cannot proceed and DNA synthesis stalls. It has been reported that human pol iota may be upregulated in a number of human cancers and specifically during the progression of breast cancer, indicating that more ..

DNA polymerases, such as Pol iota, are specifically involved in DNA repair. Pol iota specifically plays an important role in translesion synthesis, where the normal high-fidelity DNA polymerases cannot proceed and DNA synthesis stalls. It has been reported that human pol iota may be upregulated in a number of human cancers and specifically during the progression of breast cancer, indicating that pol iota's activities within normal living cells must be tightly regulated. Thus, while pol eta's (and most likely iota's) normal function is to protect humans against the deleterious consequences of DNA damage, under certain conditions they can have deleterious effects on human health. As a consequence, we propose to utilize a high throughput replication assay to identify small molecule inhibitors of pol iota.

In a collaboration between the National Institute of Child Health & Human Development (NICHD) and NIH Chemical Genomics Center, a high-throughput, fluorescent screen was developed to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR). This screen is used to identify inhibitors of pol iota.

Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol iota in columns 1, 2, and 5-48) will be dispensed into 1,536-well black solid-bottomed plate. Compounds (23 nL) will be transferred via Kalypsys pin tool equipped with 1536-pin array. The plates will then be incubated for 15 min at room temperature, and 1 muL substrate (50 nM final concentration) will be added to start the reaction and kinetically read twice at 0 min and 10 min on the Viewlux reader.

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.

Float

μM

3

Efficacy

Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.

Float

%

4

Analysis Comment

Annotation/notes on a particular compound's data or its analysis.

String

5

Curve_Description

A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.

String

6

Fit_LogAC50

The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).

Efficacy at zero concentration of compound from a fit of the data to the Hill equation.

Float

%

11

Fit_CurveClass

Numerical encoding of curve description for the fitted Hill equation.

Float

12

Excluded_Points

Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.